Post transcriptional modifications are an invariant feature of RNA molecules and the pseudouridine (Y) isomer of uracil is the most abundant modification found. Although structural studies suggest that Y contributes to the stability of RNAs, the importance of this and other potential roles in vivo remain largely unknown. In the ribosomal (r) RNAs, Y is found in the critical functional centers, suggesting an important role for Y in ribosome function. Consistent with this, rRNA pseudouridylation is essential in eukaryotes. In contrast, it is dispensable in a model archaeon while the PI?s laboratory has demonstrated that in E. coli, loss of all rRNA Y modifications has only modest effects on growth, ribosome function and all other tested phenotypes. The location and high conservation of Y modifications in rRNAs contrasts with their dispensability in model bacteria and archaea and provides the impetus for us to determine Y function. The long-range goal of the proposal is to determine the function of Y in ribosomes of diverse bacteria. This goal will be pursued in 3 complementary Specific Aims: Specific Aim 1 will use gene interaction approaches to uncover the functions of Y in E. coli. Preliminary results link Y with ribosome biogenesis and turnover, and these findings will be pursued by examining rRNA metabolism, ribosome assembly and translation activity in Y-deficient cells. Specific Aim 2 will examine two of the known effects of Y loss in bacteria, a decreased efficiency of translation termination and sensitivity to hyperosmotic stress. Genetic and physiological analyses will be used to examine these two consequences of Y loss in wild type and Y-deficient cells. Specific Aim 3 will determine if the E. coli result can be extended to other bacteria by testing if Y is also dispensable in Thermus thermophilus, a thermophilic bacterium, by deleting the Y synthases and characterizing the resulting mutants. The health relevance of the proposal derives from the association of impaired pseudouridylation with dyskeratosis congenita, a complex bone marrow failure syndrome in humans, as well as the contribution of Y to the toxicity of 5-fluorouracil, an anti-cancer therapy and the involvement of Y in rRNA folding and ribosome assembly. Given the differing routes for Y incorporation into rRNA in bacteria and eukarya, elucidation of the role of Y in ribosome biogenesis may facilitate the development of antibacterials that inhibit this essential bacterial function.